Abstract GAS is a preeminent Gram+ bacterial pathogen causing a wide spectrum of diseases in the human host. While the common outcome of a GAS encounter is asymptomatic colonization or self-limited mucosal infection, the propensity of particular GAS strains to produce systemic infection in otherwise healthy individuals defines a capacity to resist host innate immune clearance mechanisms that normally function to prevent dissemination beyond epithelial surfaces. A clone of the GAS M1T1 serotype has spread globally over the last 30 years as the leading cause of invasive infections including necrotizing fasciitis. Our laboratory has adopted a multifaceted approach to understanding those GAS and host factors that explain the diverse outcomes of this important host-pathogen interaction, using the invasive M1T1 GAS clone as a model. Our methodology has coupled precise, targeted mutagenesis and heterologous expression of candidate virulence factor genes with in vitro, ex vivo and in vivo models of disease pathogenesis, including WT, knockout and human transgenic mouse lines. We hypothesize that the outcome of GAS infection is dictated by the action and regulation of these GAS virulence factors in response to selective pressures exerted by host innate immunity. In this proposal, we will define the repertoire of bacterial virulence factors that promote the shift of GAS M1T1 strains to an invasive disease phenotype in response to innate immune selection. In Aim 1, we will test a unique and extensive panel of isogenic M1T1 GAS virulence factor mutants for neutrophil resistance, invasive phenotype switching, and systemic infection in the humanized plasminogen mouse, defining those innate immune resistance factors necessary for systemic virulence. In parallel, we will constitutively express specific virulence factors to identify if any are sufficient to promote disease progression. In the complementary studies of Aim 2, we will use pharmacologic techniques and knockout mice to define those specific aspects of host innate immune defense that exert selective pressure on GAS M1T1 favoring the shift to invasive phenotype. In Aim 3, we will determine the contribution of specific M1T1 GAS virulence genes and invasive phenotype shifting on GAS fitness during epithelial cell interactions and mucosal colonization. In this fashion, we will identify the competing selective pressures faced by this obligate human pathogen during the different stages of its overall ecology. Finally, we will assess the robustness of our experimental model (Nat Med 2007) that acquisition of a phage ?M1T1Z encoding the DNase Sda1 was a sentinel evident in the epidemic of invasive M1T1 infection, promoting resistance to phagocytic clearance through evasion of neutrophil extracellular traps. The last Aim will be achieved by exploring ?M1T1Z transduction mechanisms, phage distribution in diverse M serotype strains in the U.S. and an area of high endemic GAS disease (the Australian Northern Territory), and studying the contribution of the phage to disease switching and invasive disease in non-M1T1 strain backgrounds. Project Narrative Group A Streptococcus (GAS) is a bacteria that is a leading cause of infections in humans of all ages, from simple strep throat to life-threatening flesh-eating infections and shock. Serious disease is an unusual outcome, as most people can acquire the GAS bacterium in their throat or on their skin without developing symptoms. We are studying the ways in which the GAS bacteria shifts from an innocent member of our normal flora to an invasive pathogen, using molecular genetic techniques, assays of immune function, and mouse models of infection.